Nobiletin is an O-methylated flavonoid found in citrus peels that have anticancer, antiviral, neuroprotective, anti-inflammatory activities and depending on the cell types exhibits both pro-or anti-apoptotic properties. We have found that nobiletin decreases oxygen consumption by bovine brain isolated mitochondria in the presence of glutamate and malate and increases in the presence of succinate. In parallel, nobiletin increases NADH oxidation, a-ketoglutarate dehydrogenase activities and through matrix substrate-level phosphorylation elevates the a-ketoglutarate-dependent production of ATP. In addition, nobiletin reduces the production of peroxides in the presence of complex I substrates and slightly enhances succinate-driven H 2 O 2 formation. Besides, nobiletin induces transient elevation of membrane potential followed by mild depolarization. Affinity purified nobiletin binding proteins revealed one major anti-NDUFV1 positive protein with 52kD and NADH: ubiquinone oxidoreductase activity. This fraction can produce peroxide that is inhibited by nobiletin. We propose that nobiletin may act as a mild "uncoupler", which through activation of a-ketoglutarate dehydrogenase (a-KGDH)-complex and acceleration of matrix substratelevel phosphorylation maintains membrane potential at an abnormal level. This switch in mitochondrial metabolism could elevate succinate-driven oxygen consumption that may underlay in both pro-and anti-apoptotic effects of nobiletin.
Recent observations have established that interruption of insulin production causes deficits in learning and memory formation. We have studied the mechanism of insulin's neuroprotective effect on primary neuronal cells and in streptozotocin (STZ)-induced diabetic rat brain. We have found that in hippocampal neuronal cells insulin increases the content of farnesylated Ras and phosphorylated form of Akt. Besides, the treatment of cells by insulin leads to the activation of mitochondrial cytochrome oxidase, which is inhibited by manumycin, a farnesyltransferase inhibitor. During experimental diabetes, the content of membrane-bound GRF1 was decreased in rat hippocampus that was correlated with the reduction in mitochondrial Ras and phosphorylated forms of Akt. This redistribution in Ras-GRF system was accompanied by the alteration in the activities of CREB, NF-kB (p65) and c-Rel transcription factors. We have proposed that hypoinsulinemia induces the inhibition of Ras signalling in the neuronal cells additionally by abnormality of Ras trafficking into mitochondria.
PurposeDuring a neuronal injury, a variety of immune cells infiltrate into the local microenvironment at the demyelination site. After the destruction of the intact myelin sheath, its major constituent myelin basic protein (MBP) dissociates from the plasma membrane and acts as a free ligand on the infiltrated immune cells. MBP exhibits charge microheterogeneity as a result of post-translational modifications, but the effect of various isomers of MBP on the activity of macrophages is not known.Materials and methodsMBP was isolated and purified from bovine brain white matter. RAW 264.7 macrophages were cultured in DMEM supplemented with heat-inactivated fetal bovine serum. For evaluation of macrophage polarization following treatment of RAW 264.7 cells with MBP charge isomers, inducible nitric oxide synthase (iNOS) expression (M1 phenotype marker) and arginase-1 expression (M2 phenotype marker) were determined in cell lysates by ELISA. To assess Rac activity, G-LISA Rac Activation Assay system was used. The expression of receptor for advanced glycation end-products (RAGE) and high mobility group box 1 (HMGB1) protein were assayed by Western blot analysis.ResultsOur results have shown that minimally modified C1 component of MBP increases the expression of arginase-1 in cells, decreases the expression of iNOS, does not change the secretion of HMGB1 protein, but significantly elevates surface expression of RAGE, and in parallel, increases the activity of small GTPase Rac. On the other hand, highly modified deiminated isomer C8-MBP increases the secretion of HMGB1 protein but does not change the expression of arginase-1 or the content of RAGE.ConclusionThese data indicate that deiminated C8 isomer of MBP tends to polarize RAW macrophages into M1 phenotypes, whereas C1 enhances the activity of M2 phenotype markers.
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